Cationic cholesteryl derivatives containing cyclic polar groups

ABSTRACT

The present invention discloses compounds which are cationic cholesteryl derivatives having a nitrogen-containing ring structure as their polar head group. These compounds are useful for delivering biologically active substances to cells and for transfecting nucleic acids into cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.09/669,031, filed on Sep. 25, 2000, now U.S. Pat. No. 6,258,792, whichis a continuation of U.S. patent application Ser. No. 08/858,166, filedon Apr. 11, 1998, now abandoned, which is a U.S. National Phase ofinternational patent application number PCT/US97/06066, filed on Apr.11, 1997, which is a continuation of patent application Ser. No.08/631,203, filed on Apr. 12, 1996, now abandoned, the disclosures ofwhich are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

This invention relates to cationic lipids and their use in deliveringbiologically active substances to cells. In particular, the inventionrelates to novel cationic cholesteryl derivatives containing cyclicpolar groups and the use of these derivatives to deliver biologicallyactive substances to cells and to transfect nucleic acids into cells.

BACKGROUND OF THE INVENTION

The development of new forms of therapeutics which use macromoleculessuch as proteins or nucleic acids as therapeutic agents has created aneed to develop new and effective means of delivering suchmacromolecules to their appropriate cellular targets. Therapeutics basedon either the use of specific polypeptide growth factors or specificgenes to replace or supplement absent or defective genes are examples oftherapeutics which may require such new delivery systems. Clinicalapplication of such therapies depends not only on efficacy of newdelivery systems but also on their safety and on the ease with which thetechnologies underlying these systems can be adapted for large scalepharmaceutical production, storage, and distribution of the therapeuticformulations.

Gene therapy has become an increasingly important mode of treatingvarious diseases. The potential for providing effective treatments, andeven cures, has stimulated an intense effort to apply this technology todiseases for which there have been no effective treatments. Recentprogress in this area has indicated that gene therapy may have asignificant impact not only on the treatment of single gene disorders,but also on other more complex diseases such as cancer.

Success of a gene therapy protocol largely depends upon the vehicle usedto deliver the gene. A variety of means exist to introduce a gene insidethe cell including physical means such as microinjection (Capecchi, M.R. Cell (1980) 22:479-485), electroporation (Pacqereau, L. et al. Anal.Biochem. (1992) 204:147-151) and particle bombardment (Yang, N.-S. etal. Proc. Natl. Acad. Sci. USA (1990) 87:9568-9572)), biological meanssuch as viruses (Ferry, N. et al. Proc. Natl. Acad. Sci. (1991)88:8377-8381) and chemical means such as calcium phosphate (Wiegler, M.et al. Cell (1977) 11:223-232), DEAE dextran (Ishikawa, Y. et al. Nucl.Acid Res. (1992) 20:4367-4370), polylysine (Wu, G. Y. et al. J. Biol.Chem. (1988) 263:4429-4432) and cationic liposomes (Felgner, P. L. etal. Proc. Natl. Acad. Sci. (1987) 84:7413-7417)). Clinical applicationof such therapies depends not only on the efficacy of new deliverysystems but also on their safety and on the ease with which thetechnologies underlying these systems can be adapted for large scalepharmaceutical production, storage, and distribution of the therapeuticformulations. Thus, an ideal vehicle for the delivery of exogenous genesinto cells and tissues should be highly efficient in nucleic aciddelivery, safe to use, easy to produce in large quantity and havesufficient stability to be practicable as a pharmaceutical.

Non-viral vehicles, which are represented mainly by cationic lipids, areone type of vehicle which have, for the following reasons, beenconsidered for use in gene therapy. First, the plasmid DNA required forliposome-mediated gene therapy can be widely and routinely prepared on alarge scale and is simpler and carries less risk than the use of viralvectors such as retroviruses. Second, cationic lipids are less toxic andless immunogenic than viral vectors and the DNA complexed with thelipids is better protected from degradation by nucleases. Third,liposome-mediated gene delivery, unlike retroviral-mediated genedelivery, can deliver either RNA or DNA. Thus, DNA, RNA or anoligonucleotide can be introduced directly into cells using cationicliposomes.

Among the numerous cationic amphiphiles which have been referred to asuseful for transfecting nucleic acids into cells are cationicderivatives of cholesterol. For example, cholesterol(4′-trimethylammonio) butanoate (ChOTB) contains a trimethylammoniumgroup connected to the 3′-hydroxyl group of cholesterol via a butanoylspacer arm and cholesterol hemisuccinate choline ester (ChOSC) containsa choline moiety connected to the 3′-hydroxyl group via a succinylspacer arm. However, the transfection activities of these amphiphilesare generally weak. (Leventis, R. et al. (1989) Biochim. Biophys. Acta.,1023:124-132).

Epand et al. (U.S. Pat. No. 5,283,185) describe cationic derivatives ofcholesterol in which primary, secondary-tertiary or quaternary alkylammonium groups are linked to cholesterol via the 3-hydroxy group. Thesecationic cholesterol derivatives, including3β[N-(N′,N′-dimethylaminoethane) carbamoyl] cholesterol (DC-Chol), aredisclosed to be useful in transfecting nucleic acids into cells.

SUMMARY OF THE INVENTION

This invention relates to novel compounds having the formula:

X—R₂—R₁—O-Cholesteryl,

or salts thereof, where

R1 is a linker bond

R₂ is a direct bond, a C₁-C₁₀ linear or branched chain alkylene oralkenylene group, a C₃-C₇ cyclolalkylene, preferably C₅-C₆, or aphenylene; and X is a 4-7-membered nitrogen-containing cyclic structurewherein said cyclic structure can optionally include further heteroatomssuch as S, O or NR₃. The X-moiety can be linked to the R₂ spacer eithervia a carbon atom on the nitrogen-containing cyclic structure or via anitrogen atom of the cyclic structure. R₃ mentioned hereinabove is H,CH₃, C₂H₅, CH₂(CH₂)_(Z)SH or CH₂(CH₂)_(z) OH where z=0−3.

The invention further relates to lipid dispersions which comprise atleast one compound of the present invention where by “lipid dispersions”as used throughout the specification and claims is meant liposomes,micelles, emulsions or lipoproteins.

The present invention also relates to biologically active substance:lipid complexes formed by mixing a biologically active substance withlipid dispersions comprising at least one compound of the invention. Theinvention further relates to biologically activesubstance:lipid:polycation complexes formed by mixing a biologicallyactive substance with polycation and with lipid dispersions comprisingat least one compound of the invention.

The invention therefore provides methods for delivering biologicallyactive substances to cells where such methods may be utilized to deliversubstances to cells in vitro or in vivo by contacting cells with thecomplexes of this invention.

The invention also provides a method of transfecting cells comprising(a) mixing nucleic acid which encodes a protein or peptide or, whicheffects gene expression, with lipid dispersions comprising at least onecompound of the invention and optionally, polycation, to form nucleicacid:lipid or nucleic acid:lipid:polycation complexes and (o) contactingthe cells with the complex. It is contemplated that the methods oftransfection may be utilized in vitro or in vivo.

The invention further provides pharmaceutical compositions comprising atleast one compound of the invention; pharmaceutical compositionscomprising a lipid dispersion containing at least one compound of theinvention; and pharmaceutical compositions comprising at least onecomplex of the invention.

The invention further relates to a kit containing a compound of theinvention and/or a lipid dispersion containing at least one suchcompound. The invention also provides kits containing the complexes ofthe invention.

DESCRIPTION OF FIGURES

FIG. 1 shows the general route of synthesis for lipids 2-7. IM and PY.

FIG. 2 shows the structures of lipids 2-7 synthesized by the route shownin FIG. 1.

Lipid 2, 4{N-2-ethylamino [(3′-β-cholesteryl) carbamoyl]} piperazine;

Lipid 3, {N-2-ethylamino [(3′-β-cholesteryl) carbamoyl]} morpholine;

Lipid 4, {N-2-propylamino [(3′-β-cholesteryl) carbamoyl]} morpholine;

Lipid 5, N-methyl {4-N-amino [(3′-β-cholesteryl) carbamoyl]} piperazine;

Lipid 6, {N-2-ethylamino [(3′-β-cholesteryl) carbamoyl]} pyrrolidine;and

Lipid 7, {N-2-ethylamino [(3′-β-cholesteryl) carbamoyl]} piperidine.

FIG. 3 shows structures of (imidazole)propyl carbamoyl cholesteryl (IM)and pyridyl (ethyl) carbamoyl cholesteryl (PY), two cholesterolderivatives which contain unsaturated nitrogen-containing rings.

FIG. 4 shows luciferase activity in CHO cells transfected with pCMV-LucDNA complexed with varying nmol amounts of liposomes comprising DOPE andDC-Chol or DOPE and lipids 4 or 5 (mol:mol ratio of DOPE:cationic lipidof 1:1). Luciferase activity is indicated on the vertical axis of FIGS.4-8 as relative light units (RLU)/μg protein and “nmol lipid” on thehorizontal axis of FIGS. 4-8 refers to the total amount of liposomallipid which is mixed with DNA to form the DNA lipid complexes which usedto transfect the cells.

FIG. 5 shows luciferase activity in CHO cells transfected with pCMV-LucDNA complexed with varying nmol amounts of liposomes comprising DOPE andDC-Chol or DOPE and lipids 2, 3, 6 or 7 (mol:mol ratio of DOPE:lipid of1:1).

FIG. 6 shows luciferase activity in CHO cells transfected with pCMV-LucDNA complexed with varying nmol amounts of liposomes comprising DC-Choland DOPE or DOPE and (imidazole) propyl carbamoyl cholesteryl (IM) orDOPE and pyridyl (ethyl) carbamoyl cholesteryl (PY) (mol:mol ratio ofDOPE:lipid of 1:1).

FIG. 7 shows luciferase activity in 293 cells transfected with pCMV-LucDNA complexed with varying nmol amounts of liposomes comprising DOPE andDC-Chol or DOPE and lipid 5 in a mol:mol ratio of 1:1.

FIG. 8 shows luciferase activity in BL6 cells transfected with pCMV-LucDNA complexed with varying nmol amounts of liposomes comprising DOPE andDC-Chol or DOPE and lipid 5 in a mol:mol ratio of 1:1.

FIG. 9 shows the toxicity to CHO cells of varying nmol amounts ofliposomes (indicated as nmol lipid on horizontal axis) comprising DOPEand lipid 5 or DOPE and DC-Chol in a mol:mol ratio of 1:1: Toxicity wasassayed by measuring the amount of protein remaining in each well 36hours after treatment with the indicated amount of liposome.

FIG. 10 shows the toxicity to BL6 cells of varying nmol amounts ofliposomes (indicated as nmol lipid on horizontal axis) comprising DOPEand lipid 5 or DOPE and DC-Chol in a mol:mol ratio of 1:1. Toxicity wasassayed by measuring the amount of protein remaining in each well 36hours after treatment with the indicated amount of liposome.

FIG. 11 shows the transfection activities of liposomes formulated withvarying mol amounts of lipid 5 and DOPE (mol/mol). CHO cells weretransfected with pCMV-Luc DNA complexed with varying nmol amounts ofliposomes (horizontal axis) of the indicated lipid 5: DOPE mol/molratios and luciferase activity (vertical axis) was measured 36 hoursafter transfection with the DNA:lipid complexes.

FIG. 12 shows luciferase activity in CHO cells transfected in thepresence of 0, 10 or 20% fetal bovine serum with pCMV-Luc DNA (1 μg)complexed with 5, 10 or 20 nmol of lipid 5 in a 1:1 ratio with DOPE(mol/mol).

DESCRIPTION OF INVENTION

The present invention relates to compounds having the formula:

X—R₂—R₁—O-Cholesteryl,

or salts thereof, where the oxygen is joined directly to the 3-carbon ofthe cholesteryl molecule;

R₁ is

R₂ is a spacer arm and X is a nitrogen-containing cyclic structure.

In a preferred embodiment, R₁ is an

linker bond such that R₁—O— is

The R₂ spacer is a direct bond, a branched or linear alkylene oralkenylene chain of about 1 to about 10 carbon atoms in length, a C₃-C₇cycloalkylene, preferably a C₅-C₆ cycloalkylene, or a phenylene. Thebranched chain alkylene and alkenylene structures have alkyl side groupswhich are generally, methyl, ethyl, propyl and isopropyl groups. Thecyclic nitrogen-containing structure may be quaternized to form a polarhead group. Variations in the length of the R₂ spacer may be made toplace the positive charge of the polar head groups of the compounds incloser proximity to the negative charges of biologically activesubstances such as nucleic acids. In a preferred embodiment, R₂ is alinear alkylene group; in a more preferred embodiment, R₂ is a directbond or a linear C₁-C₃ alkylene group and in a most preferredembodiment, R₂ is a direct bond.

X is a 4-7-membered nitrogen-containing cyclic structure wherein saidcyclic structure can optionally include further heteroatoms such as S, Oor NR₃. The X moiety can be linked to the R₂ spacer either via a carbonatom on the nitrogen-containing cyclic structure or via a nitrogen atomof the cyclic structure. R₃ mentioned hereinabove is —H, —CH₃, —C₂H₅,—CH₂(CH₂)_(z)OH or —CH₂ (CH₂)_(z)SH where Z may be 0-3. Thenitrogen-containing ring may be saturated or it may contain unsaturationprovided that at least one nitrogen atom does not participate in adouble bond and is available to possess a positive charge. In apreferred embodiment, X is a 4-6 membered saturated nitrogen-containingring which optionally contains at least one additional O, S, or Nheteroatom; in a more preferred embodiment, X is a 5-6 memberedsaturated nitrogen-containing ring which optionally contains at leastone additional N, O, or S heteroatom; and in a most preferredembodiment, X is a six-membered saturated ring which optionally containsat least one additional heteroatom selected from the group consisting ofN, S or O.

Preferred embodiments of the compounds of the invention include thefollowing compounds:

1) X—R₂—R₁—O-Cholesteryl, where R₁ is

 R₂ is a direct bond or a linear C₁-C₃ alkylene chain and X is a 4-6membered nitrogen-containing cyclic structure which includes as afurther heteroatom NR₃, where R₃ is as defined above.

2) X—R₂—R₁—O-Cholesteryl where R₁ is

 R₂ is a linear C₁-C₃ alkylene chain and X is a 4-6 memberednitrogen-containing cyclic structure which contains an O or an S as anadditional heteroatom,

3) X—R₂—R₁—O-Cholesteryl where R₁ is

 R₂ is a linear C₁-C₃ alkylene chain and X is a 4-6 memberednitrogen-containing cyclic structure which contains as an additionalheteroatom NR₃, where R₃ is as defined above.

4) X—R₂—R₁—O-Cholesteryl where R₁ is

 R₂ is a direct bond and X is a 5-7 membered nitrogen-containing cyclicstructure which contains as an additional heteroatom NR₃, where R₃ is asdefined above.

In a more preferred embodiment, the compounds of the invention have thestructures 4-7 shown in FIG. 2 and in a most preferred embodiment, thecompound has structure 5 shown in FIG. 2.

The compounds of the present invention may be synthesized using standardreaction sequences known to those of ordinary skill in the art. In oneembodiment, the compounds may be synthesized by attaching an X—R₂—R₁—molecule to O-cholesteryl. In an alternative embodiment, one may buildfrom the O-cholesteryl molecule in a step-wise fashion to produce, forexample, R₁—O-Cholesteryl, then R₂—R₁—O-Cholesteryl, and thenX—R₂—R₁—O-Cholesteryl.

In yet another embodiment, the compounds of the invention may besynthesized by forming blocks of molecules and then coupling the blocksto each other to produce the desired compounds. Of course, regardless ofthe reaction sequence chosen to synthesize the compounds of theinvention, those of ordinary skill in the art will readily understandthat reactions for formation of the various functional group linkagescontained in such compound-are well known in the art (Morrison and Boyd(1979) Organic Chemistry 3rd Edition, Allyn and Bacon, Inc., Boston,Mass.).

Once synthesized, the compounds of the invention may be used toformulate lipid dispersions such as liposomes, micelles, emulsions andlipoproteins by methods known to those of ordinary skill in the art.When used to formulate liposomes, for example, the compounds of theinvention may be used in combination with other cationic lipids, neutralphospholipids or negatively charged lipids to form liposomes. Suitablecationic lipid species which may be combined with the compounds of theinvention include, but are not limited to, 1,2bis(oleoyloxy)-3-(trimethylammonio)propane (DOTAP);N-[1,-(2,3-dioleoyloxy) propyl]-N,N,N-trimethyl ammonium chloride(DOTMA) or other N-(N,N-1-dialkoxy)-alklyl-N,N,N-trisubstituted ammoniumsurfactants; 1,2 dioleoyl-3-(4′-trimethylammonio) butanoyl-sn-glycerol(DOBT) or cholesterol (4′-trimethylammonia) butanoate (ChOTB) where thetrimethylammonium group is connected via a butanoyl spacer arm to eitherthe double chain (for DOTB) or cholesterol group (for ChOTB); DORI(DL-1,2-dioleoyl-3-dimethylaminopropyl-B-hydroxyethylammonium) or DORIE(DL-1,2-O-dioleoyl-3-dimethylaminopropyl-β-hydroxyethylammonium) (DORIE)or analogs thereof as disclosed in WO 93/03709;1,2-dioleoyl-3-succinyl-sn-glycerol choline ester (DOSC); cholesterolhemisuccinate ester (ChOSC); lipopolyamines such asdoctadecylamidoglycylspermine (DOGS) and dipalmitoylphosphatidyesthanolamidospermine (DPPES), or the cationic lipidsdisclosed in U.S. Pat. No. 5,283,185,cholesterol-3β-carboxyamido-ethylenetrimethylammonium iodide,1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesterol carboxylateiodide, cholesterol-3β-carboxyamidoethyleneamine,cholesterol-3β-oxysuccinamidoethylenetrimethylammonium iodide,1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesterol-3β-oxysuccinateiodide, 2-[(2-trimethylammonio)-ethylmethylamino]ethyl-cholesterol-3β-oxysuccinate iodide,3β[N-(N′,N′-dimethylaminoethane)-carbamoyl]-cholesterol (DC-chol), and3β-[N-(polyethyleneimine)-carbamoyl]cholesterol.

Examples of preferred cationic lipids includecholesterol-3β-carboxyamidoethylenetrimethylanimonium iodide,1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesterol carboxylateiodide, cholesterol-3β-carboxyamidoethyleneamine,cholesterol-3β-oxysuccin-amidoethylenetrimethylammonium iodide,1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesterol-3β-oxysuccinateiodide,2-[(2-trimethylammonio)ethylmethylamino]-ethyl-cholesterol-3β-oxysuccinateiodide, 3β[N-(N′,N′dimethyl-aminoethane)-carbamoyl]-cholesterol(DC-chol), and 3β[N-(N′,N′-dimethylaminoethane)-carbamoyl]-cholesterol.

In addition to cationic lipids, the liposomes may also contain otherlipids. These lipids include, but are not limited to, lyso lipids ofwhich lysophosphatidylcholine (1-oleoyllysophosphatidycholine) is anexample, cholesterol, or neutral phospholipids including dioleoylphosphatidyl ethanolamine (DOPE) or dioleoyl phosphatidylcholine (DOPC).The liposomes may also contain negatively charged lipids so long as thenet charge of the complexes formed is positive. Negatively chargedlipids of the invention are those comprising at least one lipid specieshaving a net negative charge at or near physiological pH or combinationsof these. Suitable negatively charged lipid species include, but are notlimited to, phosphatidyl glycerol and phosphatidic acid or a similarphospholipid analog.

When a compound of the invention is to be combined with another lipid toformulate liposomes, a preferred lipid is a neutral phospholipid, mostpreferably DOPE. Preferred mol/mol ratios of compound of theinvention:DOPE may range from about 3:7 to about 7:3.

It is to be understood that in considering lipids which may be combinedwith the compounds of the invention to produce liposomes, those ofordinary skill in the art are not restricted only to the use of thelipids recited above but rather, any lipid composition may be used solong as a cationic liposome is produced.

Methods for producing such liposomes are known to those of ordinaryskill in the art. A review of methodologies of liposome preparation maybe found in Liposome Technology (CFC Press NY 1984); Liposomes by Ortro(Marcel Schher, 1987); Methods Biochem Anol. 33:337-462 (1988) and U.S.Pat. No. 5,283,185. Such methods include freeze-thaw extrusion andsonication. It is contemplated that both unilamellar liposomes (lessthan about 200 nm in average diameter) and multilamellar liposomes(greater than about 300 nm in average diameter) may be produced.

Once produced, the lipid dispersions of the invention may be mixed withbiologically active substances to produce a biologically activesubstance:lipid complex.

In an alternative embodiment, lipid dispersions containing a compound ofthe invention may be mixed with polycation and a biologically activesubstance to form a lipid:polycation:biologically active substancescomplex. (Gao, X et al (1996) Biochemistry 35:1027-1036). Suitablepolycations for use in forming such complexes are natural or syntheticamino acids, peptides, proteins, polyamines, carbohydrates and anysynthetic cationic polymers. Nonlimiting examples of polycations includepolyarginine, polyornithine, protamines and polylysine, polybrene(hexadimethrine bromide), histone, cationic dendrimer, spermine,spermidine and synthetic polypeptides derived from SV40 large T antigenwhich has excess positive charges and represents a nuclear localizationsignal. A preferred polycation is poly-L-lysine (PLL).

By “biologically active substances” as used throughout the specificationand claims is meant a molecule, compound, or composition, which, whenpresent in an effective amount, reacts with and/or affects living cellsand organisms. It is to be understood that depending on the nature ofthe active substance, the active substance may either be active at thecell surface or produce its activity, such as with DNA or RNA, afterbeing introduced into the cell.

Examples of biologically active substances include, but are not limitedto, nucleic acids such as DNA, cDNA, RNA (full length mRNA, ribozymes,antisense RNA, decoys), oligodeoxynucleotides (phosphodiesters,phosphothioates, phosphoramidites, and all other chemicalmodifications), oligonucleotide (phosphodiesters, etc.) or linear andclosed circular plasmid DNA; carbohydrates; antibodies, proteins andpeptides, including recombinant proteins such as for example cytokines(e.g. NGF, G-CSF, GM-CSF), enzymes, vaccines (e.g. HBsAg, gp120);vitamins, prostaglandins, drugs such as local anesthetics (e.g.procaine), antimalarial agents (e.g. chloroquine), compounds which needto cross the blood-brain barrier such as anti-parkinson agents (e.g.leva-DOPA), adrenergic receptor antagonists (e.g. propanolol),anti-neoplastic agents (e.g. doxorubicin), antihistamines, biogenicamines (e.g. dopamine), antidepressants (e.g. desipramine),anticholinergics (e.g. atropine), antiarrhythmics (e.g. quinidine),antiemetics (e.g. chloroprimamine) and analgesics (e.g. codeine,morphine) or small molecular weight drugs such as cisplatin whichenhance transfection activity, or prolong the life time of DNA in andoutside the cells.

When the biologically active substances is an antigenic protein orpeptide, the complexes formed by mixing the protein or peptide withlipid dispersions containing compound(s) of the present invention may beutilized as vaccines.

Preferred biologically active substances are negatively chargedsubstances such as nucleic acids, negatively charged proteins andcarbohydrates including polysaccharides, or negatively charged drugs.

The present invention therefore provides methods for deliveringbiologically active substances to cells. In one embodiment the methodcomprises:

(a) mixing a biologically active substance with a lipid dispersioncontaining at least one compound of the invention to form a biologicallyactive substance:lipid complex; and

(b) contacting the cells with the complexes.

In an alternative embodiment, the method comprises:

(a) mixing a biologically active substance with lipid dispersions and apolycation to form a biologically active substance:lipid:polycationcomplex; and

(b) contacting the cells with the complex.

It is contemplated that the methods of the invention may be used todeliver biologically active substances to cells in vitro or in vivo.

It is further understood that when the complexes of the invention arecontacted with cells in vivo, the complexes may be used therapeuticallyand/or prophylactically depending on the biologically active substancecontained in the complexes. The invention therefore provides fortherapeutic and/or prophylactic formulations comprising the complexes ofthe invention where such formulations comprise the complexes in aphysiologically compatible buffer such as, for example, phosphatebuffered saline, isotonic saline or low ionic strength buffer such as10% sucrose in H₂O (pH 7.4-7.6) or in Hepes (pH 7-8, a more preferred pHbeing 7.4-7.6). The complexes may be administered as aerosols or asliquid solutions for intratumor, intravenous, intratracheal,intraperitoneal, and intramuscular administration. Those of ordinaryskill in the art would readily understand that the actual amount ofcomplex to be administered will depend upon the route of administration,the pharmaceutical properties of the individual treated, as well as theresults desired.

The present invention also provides methods for transfecting nucleicacids into cells in vitro or in vivo. It is to be understood that whenused to transfect cells in vivo the methods of transfection may be usedfor gene therapy. It is also contemplated that when used to formulatenucleic acid:lipid and nucleic acid:lipid polycation complexes usefulfor transfecting cells, the lipid dispersions can contain a compound ofthe invention or a compound having the formula:

X—R₂—R₁-Cholesteryl

where

R₁ is a direct bond to the 3 carbon of cholesteryl or a linker bond ofthe formulae

R₂ is a direct bond, a C₁-C₁₀ linear or branched chain alkylene oralkenylene group, a C₃-C₇ cycloalkylene, preferably C₅-C₆ cycloalkylene,or a phenylene, and X is a 4-7-membered nitrogen-containing cyclicstructure wherein said cyclic structure can optionally include furtherheteroatoms such as S, O or NR₃. The X moiety can be linked to the R₂spacer either via a carbon atom on the nitrogen-containing cyclicstructure or via a nitrogen atom of the cyclic structure. R₃ mentionedhereinabove is H, CH₃, C₂H₅, CH₂(CH₂)_(z)SH, or CH₂(CH₂)_(z)OH wherez=0−3.

These compounds and the compounds of the invention preferably contain asaturated nitrogen-containing cyclic structure when used to formcomplexes for transfecting nucleic acids into cells.

In one embodiment, the method for transfecting nucleic acids into cellscomprises:

(a) mixing nucleic acids with lipid dispersion and optionally polycationto form a nucleic acid:lipid or nucleic acid:lipid:polycation complex;and

(b) contacting cells with the complexes.

Nucleic acids to be transfected by the above method are nucleic acidswhich encode proteins or peptides or which regulate gene expression byeffecting transcription and/or translation.

The ability of a compound to transfect nucleic acids into cells may betested by contacting cells to complexes formed between a plasmidconstruct containing a reporter gene and lipid dispersions comprising atleast one such compound. Such reporter genes are known to those ofordinary skill in the art and include, but are not limited to, thechloramphenicol acetyltransferase gene, the luciferase gene, theβ-galactosidase gene and the human growth hormone gene. Cells which maybe transfected by the complexes includes those cells which may betransfected by prior art lipid dispersions.

Any articles or patents referenced herein are hereby incorporated byreference. The following examples illustrate various aspects of theinvention but are intended in no way to limit the scope thereof.

EXAMPLES

Materials and Methods

Synthesis of Cholesteryl Derivatives 2-7, IM and PY

In a 100 ml round-bottomed flask equipped with a stirrer and a droppingfunnel, 1.1 mmol of reagent R—NH₂ and 0.05 ml triethylamine weredissolved in 15 ml dry chloroform. Cholesterol chloroformate (1 mmol) in15 ml dry chloroform was added dropwise to the reagent solution with aconstant stirring. The reaction was allowed to stir for 30 more minutesafter the complete addition of cholesterol chloroformate. The thin layerchromatography (TLC) analysis of the reaction using silica gel TLCplates (7.5×2.5 cm) containing F₂₅₆ florescent indicator indicated thatthe reaction was complete. The solvent was evaporated to dryness and thesolid residue was washed extensively with cold acetonitrile.

Purity of the product was judged by TLC analysis. When needed, furtherpurification was performed on silica gel column usingchloroform-methanol (75:25 vol/vol) as the eluent.

All the compounds were white amorphous powder. The yields for compounds2-7, IM and PY varied between 74-85%.

Formulation of Liposomes

2 μmol of lipid dissolved in chloroform was transferred to a test tube.A solution of DOPE (2 umol) in chloroform was then added to this testtube. The chloroform was evaporated by passing a stream of nitrogenthrough the test tube to obtain a thin film of at the bottom of the testtube. Further drying was done in a desiccator under high vacuum for 30minutes. 2 ml of distilled water was then added to the test tubes. Thetest tubes were stoppered and vortexed for one minute followed bysonication until the size of the particles was between 100 to 200 nm.The size of the particles was measured by Coulter particle sizeanalyzer.

Cell Lines

CHO, 293 and BL6 cells were obtained from the American Type CultureCollection (ATCC). Chinese Hamster Ovarian (“CHO”) cells were maintainedin F-12 nutrient medium supplemented with 10% fetal bovine serum,antibiotics (1% penicillin-streptomycin) and 1% glutamine; humanembryonic kidney 293 cells and murine melanoma BL6 cells were culturedin DMEM medium supplemented with 10% fetal bovine serum, antibiotics (1%penicillin-streptomycin) and 1% glutamine.

Transfection Protocol

All the transfection experiments were performed in 48-well plates in theabsence of serum except when specifically mentioned otherwise. Each wellreceived DNA:lipid complex formed by mixing 1 μg of DNA (puc21 CMVluciferase or “pCMV-Luc”) and various amounts of liposomes formulatedfrom DOPE and the different cationic cholesterol derivatives (lipids2-7, IM and PY). The DNA:lipid complexes were incubated with cells for 5hours at 37° C. after which the cells were washed and resuspended innutrient media (media+10% serum+antibiotic+1% glutamine). Cells wereharvested 36 hours after transfection by lysis with 200 μL lysis bufferand aliquots of the lysate were assayed for luciferase activity andamount of protein.

Luciferase Activity Assay

Luciferase activity was assayed by measuring the luminescence obtainedafter adding the substrate (Promega) to in aliquot of lysate. Thisluminescence was measured in a luminometer for 25 seconds.

Assay for the Toxicity of Lipids

The toxicity of lipids was assayed by measuring the proteinconcentration in each well by Coomassie blue reagent (Pierce) 36 hoursafter transfection. Greater concentrations of protein per well indicatedless toxicity of the tested lipids.

Example 1 Comparison of the Transfection Activities in CHO Cells ofDNA:lipid Complexes Formulated by Mixing DNA and Liposomes ContainingDOPE and DC-Chol or DOPE and Lipids 2-7

FIGS. 4 and 5 show the luciferase activity in CHO cells of DNA:lipidcomplexes formulated using DC-Chol:DOPE liposomes or lipids 2, 3, 4, 5,6 or 7. DOPE liposomes. The results presented in FIG. 4 show that theorder of activity is lipid 5>DC-Chol>lipid 4; while FIG. 5 shows thatlipids 6 and 7 are less active than DC-Chol and that lipids 2 and 3 havelow activity. In sum, the results presented in FIGS. 4 and 5 suggest thefollowing conclusions regarding the structure-activity relationship oflipids 2-7.

First, compounds having nitrogen in the ring can be equally or moreactive than those without ring nitrogens. The bulk of the ring does notinterfere with the binding of the DNA. Condensation studies performedwith these lipids and DNA corroborated this conclusion (data not shown).Second, the high activity of lipid 5 indicates that the spacer groupbetween cholesterol and the charged head group can be very small. Third,the hydrogen bonding ability of oxygen does not produce any furtherenhancement in transfection activity. Fourth, the total lipid:DNA ratiofor optimal activity varied slightly for different lipids. Thedifferences in the apparent charge available for neutralization of DNAmay be responsible for this difference.

In addition, the low activity of lipid 2 as shown in FIG. 5 wassurprising since lipid 2 has a pyrazine ring and consequently it has twocharges under physiological pH. Without being bound by theory, andwithout limiting the invention to even or odd numbered rings, onepossible explanation for the low activity of lipid 2 could be thedistance between the two nitrogens since it has been shown that distancebetween two nitrogens plays an important role in condensation of DNA.(Yoshikawa et al. (1995) FEBS Lett., 361:277-281). This publicationreported that diaminoalkanes with odd number of carbon atoms compact theDNA whereas those with even number of carbon atoms do not. The lowactivity of lipid 3 in FIG. 5 is also surprising since lipid 4, whichhas only one carbon atom more than lipid 3, showed reasonable activity.The low activity of lipid 3 therefore appears to indicate that activityof the lipid might be sensitive to the spacer length.

Example 2 Comparison of Transfection Activity of DNA:Lipid ComplexesFormulated by Mixing DNA and Liposomes Containing DOPE and EitherDC-Chol or Lipid IM or PY

FIG. 6 shows that lipid IM is slightly less active than DC-Chol whilelipid PY is much less active. It is believed that the low activity oflipid PY is because the only nitrogen in the pyridine ring is in adouble bond and is therefore unavailable to possess a positive chargefor interaction with the negatively charged DNA.

Example 3 Comparison of Transfection Activity of DNA:Lipid ComplexesFormulated by Mixing DNA and Liposomes Containing DOPE and Either Lipid5 or DC-Chol

Lipid 5 was further compared with DC-Chol by transfecting BL6 cells withlipid 5- or DC-Chol-containing DNA:lipid complexes (FIGS. 7 and 8respectively). The results presented in FIGS. 7 and 8 show that lipid 5is almost equally as active as DC-Chol in both 293 (FIG. 7) and BL6(FIG. 8) cells. Lipid 5 was also tested in several cell lines includingHeLa, Siha and Caski which are somewhat difficult to transfect whencompared with CHO or 293 cells. In all these cell lines, lipid 5 wasfound to be slightly better than DC-Chol in its ability to transfectcells (data not shown). All the lipids showed absolute requirement ofDOPE as the helper lipid and little or no activity was seen in theabsence of DOPE (data not shown).

Example 4 Toxicity of Lipid 5 Relative to DC-Chol

FIGS. 9 and 10 show the toxicity of lipid 5 compared to that of DC-Cholin CHO and BL6 cells respectively, where toxicity was assessed bymeasuring the amount of protein in each well after transfection of cellswith DNA:lipid complexes formulated by mixing DNA and liposomescontaining DOPE and DC-Chol or DOPE and lipids. The results show thatcells transfected with lipid 5-containing complex have more protein thancells transfected with DC-Chol-containing complex. In addition, whenobserved under microscope, the cells transfected with lipid 5-containingcomplex were healthy whereas those transfected with DC-Chol-containingcomplex showed some necrosis (data not shown). This reduced toxicity oflipid 5 relative to DC-Chol is the key feature of lipid 5. Of interest,lipids 2-4, 6 and 7 were also less toxic than DC-Chol while lipid PYexhibited comparable toxicity to DC-Chol and lipid IM was more toxicthan DC-Chol.

Example 5 Optimization of Ratio Between Lipid 5 and DOPE

FIG. 11 shows transfection activities of DNA:lipid complexes formulatedusing by mixing 1 μg of DNA and varying nmol amounts of liposomescontaining different mol:mol ratios of lipid 5 and DOPE. The X axis ofFIG. 11 refers to the total liposomal lipid mixed with 1 μg pCMV-LucDNA:lipid complexes. All the ratios of lipid 5:DOPE shown formed stableliposomes (as measured by retention of diameter over time) but thecomplex formed from liposomes having a lipid 5:DOPE ratio of 6:4 showedthe highest activity among all complexes while DNA:lipid complexesformed from-liposomes having lipid 5:DOPE ratios of 5:5 and 4:6 had verysimilar transfection activities. Complexes formulated from liposomeshaving ratios outside these ratios (for example, lipid 5: DOPE ratios of2:8 or 8:2) showed less activity indicating that liposomes containingroughly equal mol amounts of both lipid 5 and DOPE form more activecomplexes.

Example 6 Serum Sensitivity of Lipid 5

Serum sensitivity of lipid 5 was tested by adding fetal bovine serum toDNA:lipid complexes formed by mixing pCMV-Luc DNA and liposomes (1:1lipid 5:DOPE) The serum concentrations were then adjusted to 10% or 20%of the final volume. The DNA:lipid complexes containing serum were thenused to transfect CHO cells. Three different liposomal lipid:DNA ratioswere chosen to observe the effect of the serum on the charge of theDNA:lipid complex. For example, at a ratio of 5 nmol lipids to 1 μg DNA,the complex has excess DNA and should be negatively charged. At 1 μgDNA:10 nmol lipid 5, the DNA:lipid complex should be slightly positiveand at 1 μg DNA:20 nmol lipid 5 the DNA:lipid complex has considerableamount of positive charge. FIG. 12 shows the results of thesetransfections where the data indicates that at lower ratios of lipid 5to DNA the activity of the lipid 5 is affected by the serum to a lesserextent. For example, approximately 40% of the activity was retainedrelative to the activity of the 0% serum complexes when 5 and 10 nmollipid 5 in the presence of 10% serum were used to transfect cells.However, when 20 nmol of lipid 5 in the presence of 10% serum was usedto transfect cells, only 10% activity was retained indicating thesensitivity of the lipid to serum. DNA:lipid complexes formed fromliposomes containing DOPE and either DC-Chol or DOTMA both showedsimilar serum sensitivity (data not shown)

Example 7 In Vivo Transfection Using DNA:Lipid Complexes Formulated byMixing pCMV-Luc DNA and Lipid 5: DOPE Liposomes

Mice are injected intratumorally or intravenously with 50 μl of solutioncontaining pCMV-Luc DNA (30 μg) complexed with 30 nmols of liposomallipid (1:1 mol/mol lipid 5: DOPE). Animals are sacrificed 1-5 days laterand extracts from tumor tissue (intratumor injection) or lung, liver andblood (intravenous injection) are assayed for luciferase activity andprotein levels. The results show that DNA:lipid complexes containinglipid 5 exhibit in vivo transfection activity.

What is claimed is:
 1. A method for transfecting nucleic acids intocells, the method comprising: contacting said cells with a nucleicacid:lipid complex, wherein the complex comprises a nucleic acid and acompound having the structure: X—R₂—R₁—O-Cholesteryl, wherein R₁ is alinker bond

R₂ is a direct bond, a branched or linear alkylene or alkenylene chainof 1 to 10 carbons in length, a C₃-C₇ cycloalkylene, or a phenylene; Xis a saturated 4-7-membered nitrogen-containing cyclic structure; and Xis linked to the R₂ spacer via a carbon atom of the nitrogen-containingcyclic structure or via a nitrogen atom of the cyclic structure.
 2. Themethod of claim 1, wherein R₁ is


3. A method of claim 2, wherein R₁ is —C(═O)—.
 4. The method of claim 1,wherein R₂ is a direct bond or a linear C₁-C₃ alkylene chain.
 5. Themethod of claim 4, wherein X is a 5-6-membered nitrogen-containingcyclic structure.
 6. The method of claim 5, wherein X further comprisesa heteroatom selected from the group consisting of S, O, and NR₃,wherein R₃ is —H, —CH₃, —C₂H₅, —CH₂(CH₂)_(Z)OH or —CH₂(CH₂)_(Z)SH,wherein Z is 0-3.
 7. The method of claim 6, wherein R₂ is a linear C₂alkylene chain.
 8. The method of claim 5, wherein X is a 6-memberednitrogen-containing cyclic structure.
 9. The method of claim 8, whereinX further comprises a heteroatom selected from the group consisting ofS, O, and NR₃, wherein R₃ is —H, —CH₃, —C₂H₅, —CH₂(CH₂)_(Z)OH or—CH₂(CH₂)_(Z)SH, wherein Z is 0-3.
 10. The method of claim 9, wherein R₂is a direct bond or is a linear C₂ alkylene chain.
 11. The method ofclaim 10, wherein R₃ is H and R₂ is a linear C₂ alkylene chain.
 12. Themethod of claim 10, wherein R₂ is a direct bond.
 13. The method of claim12, wherein R₃ is a methyl group.
 14. The method of claim 8, wherein Xis a 6-membered nitrogen-containing cyclic structure which includes anadditional heteroatom O.
 15. The method of claim 14, wherein R₂ is alinear C₁₋₃ alkylene chain.
 16. The method of claim 1, wherein R₂ is aC₅-C₆ cycloalkylene.
 17. The method of claim 1, wherein X comprises afurther heteroatom.
 18. The method of claim 1, wherein X comprises afurther heteroatom selected from the group consisting of S, O, and NR₃,where R₃ is —H, —CH₃, —C₂H₅, —CH₂(CH₂)_(Z)OH or —CH₂(CH₂)_(Z)SH, whereinZ is 0-3.
 19. The method of claim 1, wherein the complex furthercomprises a polycation.
 20. The method of claim 19, wherein the cationis a polyamine.
 21. The method of claim 19, wherein the polycation is apoly-L-lysine.
 22. The method of claim 1, herein the nucleic acid isselected from the group consisting of a DNA and an RNA molecule.
 23. Themethod of claim 1 wherein the nucleic acid encodes a protein or peptideor regulates gene expression by effecting transcription and/ortranslation.
 24. The method claim 1, wherein the method comprisescontacting said cells with a liposome comprising said nucleic acid:lipidcomplex.
 25. The method of claim 1, wherein the complex furthercomprises a cationic lipid.
 26. The method of claim 25, wherein thecationic lipid is selected from the group consisting ofcholesterol-3β-carboxyamidoethylenetrimethylammonium iodide,1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesterol carboxylateiodide, cholesterol-3β-carboxyamidoethyleneamine,cholesterol-3β-oxysuccinamidoethylenetrimethylammonium iodide,1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesterol-3β-oxysuccinateiodide,2-[(2-trimethylammonio)ethylmethylamino]ethyl-cholesterol-3β-oxysuccinateiodide, 3β[N-(N′,N′-dimethylaminoethane)-carbamoyl]-cholesterol(DC-chol), and 3β[N-(polyethyleneimine)-carbamoyl]-cholesterol.
 27. Themethod of claim 1, wherein the complex further comprises a neutrallipid, a positively charged lipid or a negatively charged lipid.
 28. Themethod of claim 1, wherein the complex further comprises a lipidselected from the group consisting of cholesterol, dioleoylphosphatidylethanolamine and dioleoyl phosphatidyicholine.
 29. Themethod of claim 1, wherein R₁ is

R₂ is a direct bond or a linear C₁-C₃ alkylene chain, and X is a4-6-membered nitrogen-containing cyclic structure comprising a furthernitrogen heteroatom.
 30. The method of claim 1, wherein R₁ is

R₂ is a linear C₁-C₃ alkylene chain, and X is a 4-6-memberednitrogen-containing cyclic structure comprising a further heteroatomselected from the group consisting of O, S, and N.
 31. The method ofclaim 1, wherein R₁ is

R₂ is a direct bond, and X is a 5-7-membered nitrogen-containing cyclicstructure comprising a further nitrogen heteroatom.
 32. The method ofclaim 1, wherein the compound is


33. The method of claim 1, wherein, prior to contacting said cells withthe complex, the method further comprises: mixing the nucleic acid witha lipid to form the nucleic acid:lipid complex.
 34. The method of claim33, wherein the nucleic acid is selected from the group consisting of aDNA and an RNA molecule.
 35. The method of claim 33, wherein the nucleicacid encodes a protein or peptide or regulates gene expression byeffecting transcription and/or translation.
 36. The method of claim 29,30 or 31, wherein X further comprises at least one additional heteroatomNR₃ where R₃ is —H, —CH₃, —C₂H₅, —CH₂(CH₂)_(Z)OH or —CH₂(CH₂)_(Z)SH,wherein Z is 0-3.
 37. The method of claim 1, wherein the methodcomprises contacting the cells with a pharmaceutical compositioncomprising the nucleic acid:lipid complex.
 38. The method of claim 1,wherein the method comprises contacting the cells with said complex in aphysiologically acceptable carrier.
 39. The method of claim 1, whereinthe method comprises contacting said cells with a lipid dispersioncomprising said nucleic acid:lipid complex.
 40. The method of claim 1,25 or 27, wherein the cells are contacted with the complex in vivo, saidmethod comprising administering the complex to an animal or human in anamount effective to transfect the nucleic acid into the cells of theanimal or human.
 41. A method transfecting nucleic acids into cells, themethod comprising: contacting said cells with a nucleic acid:lipidcomplex, wherein the lipid comprises a compound having the structure:X—R₂—R₁-Cholesteryl, wherein R₁ is a direct bond to the 3 carbon ofcholesteryl or a linker bond

R₂ is a direct bond, a branched or linear alkylene or alkenylene chainof 1 to 10 carbons in length, a C₃-C₇ cycloalkylene, or a phenylene; Xis a 4-7-membered nitrogen-containing cyclic structure; and X is linkedto the R₂ spacer via a carbon atom of the nitrogen-containing cyclicstructure or via a nitrogen atom of the cyclic structure.
 42. A methodfor transfecting nucleic acids into cells, the method comprising:contacting said cells with a nucleic acid:lipid complex, wherein thecomplex comprises a nucleic acid and a compound having the structure:X—R₂—R₁—O-Cholesteryl, wherein

R₁ is a linker bond —NH—C—, —C—, or O—C—; R₂ is a direct bond, abranched or linear alkylene or alkenylene chain of 1 to 10 carbons inlength, a C₃-C₇ cycloalkylene, or a phenylene; X is a 4-7-memberednitrogen-containing cyclic structure; X is linked to the R₂ spacer via acarbon atom of the nitrogen-containing cyclic structure or via anitrogen atom of the cyclic structure; and X further comprises aheteroatom selected from the group consisting of S, O, and NR₃, whereinR₃ is —H, —CH₃, —C₂H₅, —CH₂(CH₂)_(Z)OH or —CH₂(CH₂)_(Z)SH, wherein Z is0-3.